PROJECT SUMMARY Cell surface inhibitory receptors (IRs) PD1 and LAG3 control cellular signaling and critical cell intrinsic effector T cell function and differentiation. Yet, these IRs are also expressed on exhausted intratumoral T cells, limiting disease clearance. IRs are critical to maintain immune control, but also represent a major barrier to effective anti-tumor immunity. PD1 and LAG3 are synergistic in multiple diseases, including cancers, and many CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs) express PD1/LAG3. Indeed, we showed that tumor immunotherapy targeting PD1/LAG3 can induce almost complete remission of tumors in mice, and clinical evidence now supports the benefit of single (anti-PD1) or dual (anti-PD1/LAG3) IR blockade. This project will test the central hypothesis that ?PD1 and LAG3 synergize to enforce tumor-induced tolerance using a combination of overlapping and unique cell intrinsic and extrinsic functions in CD4+ and CD8+ effector T cells?. Due to potency of tolerance in the tumor microenvironment (TME), mouse models represent an outstanding system to study IR mechanisms to compare with processes in autoimmune (Project 1) and chronic viral settings (Project 3). We will pursue 3 Aims: AIM 1: What are the relative and synergistic contributions of PD1 and LAG3 in limiting the initial CD8+ T cell anti-tumor effector response? We hypothesize that ?PD1 and LAG3 do not initiate but temporally re- enforce progression toward CD8+ T cell exhaustion by limiting population dynamics, differentiation, metabolic health, and polyfunctionality?. We will use a Quad AT system that co-transfers tumor-specific CD8+ T cells that are wild-type or lack PD1 and/or LAG3 for functional and transcriptional analysis in the same microenvironment. AIM 2: What are the relative and synergistic contributions of PD1 and LAG3 in limiting initial CD4+ Tconv anti-tumor effector response? We hypothesize that ?PD1/LAG3 have synergistic effects on CD4+ Tconv cells by differentially modulating their function and capacity for CD8+ T cell help?. We will interrogate the role of PD1/LAG3 on CD4+ T cells using our novel CreERT2 line to delete alleles in CD4+ Tconv but not CD8+ T cells or T regs . AIM 3: What is the impact of PD1/LAG3 on the durability of anti-tumor T cell memory and maintenance of systemic immunity? We hypothesize that ?PD1 and/or LAG3 limit durability of anti-tumor immunity by impacting Tex reinvigoration and Tconv cell memory development and maintenance, blunting their recall response and limiting systemic immunity?. We will ask 2 questions: (A) What is the impact of PD1/LAG3 loss on Tex and their reinvigoration? (B) What is the impact of PD1/LAG3 on maintenance of T cell anti-tumor memory and recall response? PPG Interactions: Project 2 will collaborate with Project 1 to compare the impact of PD1/LAG3 loss on CD4+ T cell function in autoimmune versus tumoral settings; with Project 3 to compare CD8+ T cell exhaustion induced by tumors or chronic viral infections; and with Core A to exchange data, Core B to obtain mice, Core C for transcriptional analysis and CRISPR/sgRNA sequences, and Core D for immunohistological analysis.